Immune thrombocytopenia (ITP) is the most prevalent bleeding disorder, affecting the lives of tens of millions of people worldwide. In individuals with ITP, antibodies are developed against common platelet surface proteins and cause the immune system to attack platelets and remove them from the bloodstream. ITP is most commonly treated using immunosuppressive treatments like steroids or intravenous immunoglobulin (IVIG). Unfortunately, approximately 20% of patients are refractory to these treatments, and a larger percentage only respond transiently. Resistance to IVIG treatment suggests that there is an alternative mechanism of platelet clearance which plays a role in IVIG-resistant ITP patients. One of the most abundant platelet surface proteins is the heterotetrameric GPIb-IX receptor complex. The receptor is responsible for binding to ligands such as von Willebrand factor, and leading to clearance of the platelet from the blood. There is a strong correlation between patients who are refractory to IVIG treatment and antibodies against the ligand binding domain (LBD) of GPIb-IX in patient serum. Additionally, injection of anti-LBD monoclonal antibodies (MAbs) into mice is sufficient to cause thrombocytopenia. The ability to induce thrombocytopenia makes these MAbs unique, since almost all antibodies against other parts of GPIb-IX cannot induce thrombocytopenia. Current models of GPIb-IX signaling are not sufficient to explain the unique ability of anti-ligand binding domain antibodies to induce clearance. Therefore, determining the mechanism of GPIb-IX signaling is vital for complete understanding of platelet clearance and thrombocytopenia. Recently, the mechanosensory domain (MSD) of GPIb-IX has been hown to be involved in GPIb-IX signaling. This disordered region of the complex is normally coiled proximal to the membrane and the other subunits of GPIb-IX. However, a segment of the MSD must be exposed and unfolded in order for part of the GPIb-IX complex to be ?shed?, which is an important signal for clearance. We hypothesize that antibodies against the ligand binding domain of GPIb-IX induce clearance by causing the unfolding of the MSD and perturbing inter-subunit interfaces. In specific aim 1, we will determine whether antibodies which bind the ligand-binding domain and cause clearance also unfold the MSD by analyzing clearance signals via flow cytometry, and correlating this to increased exposure of the epitope for an antibody which binds the MSD. In specific aim 2, we will investigate the role of inter-subunit interfaces in GPIb-IX signaling. We will do this by making use of mutants with altered residues in their inter-subunit interfaces, as well as antibody agonists and antagonists of receptor signaling. My proposed studies will elucidate fundamental mechanisms of GPIb-IX mediated platelet clearance and facilitate future development of novel diagnostic approaches and therapies for ITP and other thrombocytopenic conditions.